Solvent-free process for obtaining phospholipids and neutral enriched krill oils

ABSTRACT

Organic solvent-free processes for obtaining krill oil compositions are disclosed. The processes include a) cooking krill in a cooker vessel for a time and temperature sufficient to denature the protein content of the krill and cause a first solid krill fraction and first liquid krill fraction to be formed while substantially avoiding emulsification of the first solid and first liquid krill fractions; b) removing the first solid and first liquid krill fractions from the cooker vessel at a temperature of at least about 90° C.; c) separating the first solid fraction and the first liquid fraction; and d) obtaining krill oil with neutral enriched from the first liquid fraction, and e) by pressing of first solid fraction to obtain press liquid or a second liquid krill fraction for obtaining krill oil with phospholipids enriched krill oil, the separating and the obtaining steps being carried out without the use of organic solvents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. patent application Ser. No. 13/096,644,filed Apr. 28, 2011, which in turn is a Continuation-in-Part ofPCT/IB2009/007269, filed Oct. 30, 2009, the contents of each of whichare incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to processes for obtaining krill oil withpoly-unsaturated fatty acid docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA) in the phospholipids fraction, withastaxanthin, and neutral lipids coming from the meal processing line,used for human health applications while the resulting krill meal willhave a low fat content. This invention also discloses a procedure forobtaining a dried complex that contains krill oil in combination withphospholipids linked to DHA and EPA, proteins and astaxanthin for theuse on human-health human applications.

BACKGROUND ART

Krill corresponds to a group of small and abundant marine crustaceans inthe order Euphasiaceae, living in the pristine Antarctic Ocean that isconsidered the feed base of the all Antarctic eco-system. The Antarctickrill, in particular those that live at the Antarctic and sub-Antarcticregions, are composed of 11 Euphasia species, being dominant Euphausiasuperba, Dana and Euphausia. crystallorophias.

In recent years, krill has acquired great interest as a potential sourceof protein and other active biological products (Ellingsen, T. and Mohr,V. 1979. Process Bioch. 14:14; Suzuki, T. 1981. Fish and krill proteinprocessing technology. London, Applied Science Publishers). The largeexpectation ciphered in the South Antarctic krill is based in the largebiomass existent at the Antarctic oceans, estimated between 100 to 500millions tons. It has been suggested that captures of krill could reachup to 50-100 millions tons/year, quantity that is equivalent to thetotal fish capture in the world (Budzinski, E., Bykowski, P. andDutkiewicz, D. 1986. Posibilidades de elaboración y comercialización deproductos preparados a partir de krill del Antartico, FAO Doc. T,c.Pesca (268):47p).

There are several publications related to the krill lipid content andcomposition (Grantham, G. J. 1977. The Southern Ocean. The utilizationof Krill. Southern Ocean Fisheries Survey Programme, Rome. FAOGLO/SO/77/3:63p; Budzinski et al. 1986. Loc. cit.; Ellingsen and Mohr.1979. Loc. cit.). The lipid content is about 10-26% of the krill dryweight, depending on the season of the year and its sexual maturity aswell as body size. Usually the krill fatty period is between March toJune each year. The female krill has near double amount of lipids thanthe male. The lipid concentration increases with age and decreasesrapidly after the spawning. Krill lipids distribution studies showedthat lipid rich areas are located along the digestive tract, between themuscle bundles and under the exoskeleton (Saether, O., Ellingsen, T. andMohr, V. 1985. Comp. Biochem. Physiol. 81B:609).

The main krill lipid fractions include triglycerides, phospholipids, aswell as its sterols and esters (Grantham. 1977. Loc. cit.; Budzinski etal. 1986. Loc. cit.). The average content of phospholipids is about 69%and triglycerides about 26%. The krill phospholipids fraction, rich inpolyunsaturated fatty acids, particularly 20:5 and 22:6, correspond toapproximately 50% of total phospholipids.

There are several publications describing Krill lipid composition. Thefollowing being the most relevant among them:

-   1. Gordeev et al. described that E. superba contains about 5% of its    natural weight of extractable lipids, more than half of which are in    the form of phospholipids—phosphatidylcholine (33-36% of the lipids    total), phosphatidylethanolamine (15-17%), lysophosphatidylcholine    (3-4%), others (2-3%)—while triacylglycerols predominate (32-35%)    among the phosphorus-free components. In the first two phospholipids    the dominating fatty acid residue is the arachidonic acid residue    (more than 40% of the acyl residues total) and the amount of    eicosapentaenoic acid residues (C20: 5w3) are about 13 and 28%,    respectively. (Gordeev, K. Y. et al. 1990. Fatty acid composition of    the main phospholipids of the Antarctic krill Euphausia superb.    Chemistry of Natural Compounds. 26: 143-147).    -   Fricke et al. described that Euphausia superba Dana lipid        composition was phosphatidylcholine (33-36%),        phosphatidylethanolamine (5-6%), triacylglycerol (33-40%), free        fatty acids (8-16%) and sterols (1.4-1.7%). Wax esters and        sterol esters were present only in traces. More than 50 fatty        acids could be identified and the major ones being 14:0, 16:0,        16:1(n-7), 18:1(n-9), 18:1(n-7), 20:5(n-3) and 22:6(n-3).        Phytanic acid was found in a concentration of 3% of total fatty        acids. Short, medium-chain and hydroxy fatty acids (C<10) were        not detectable. The sterol fraction consisted of cholesterol,        desmosterol and 22-dehydrocholesterol. (Fricke, H. et al. 2006.        Lipid, sterol and fatty acid composition of Antarctic krill        (Euphausia superba Dana). Lipids, 19:821-827).-   2. Falk-Petersen et al described that lipids of Arctic and Antarctic    euphausiids show a seasonally-dependent high lipid content, and    neutral lipids, whether wax esters or triacylglycerols, are    primarily accumulated for reproduction. The Arctic Thysanoessa    inermis and the Antarctic Euphausia crystallorophias contain high    levels of wax esters and higher concentrations of 18:4(n-3) and    20:5(n-3) and a lower ratio of 18:1(n-9)/(n-7) fatty acids in their    neutral lipids than those in the Arctic Thysanoessa raschii and the    Antarctic Thysanoessa macrura and Euphausia superba. Large amounts    of phytol in the lipids of T. raschii and E. crystallorophias during    winter suggest the ingestion of decaying algae originated from    sedimenting material or in sea ice. Thysanoessa raschii, T. macrura,    and E. superba have a high ratio of 18:1(n-9)/(n-7) fatty acids,    indicating animal carnivory (Falk-Petersen, S. et al. 2000. Lipids,    trophic relationships, and biodiversity in Arctic and Antarctic    krill. Can. J. Fish. Aquat. Sci. 57: 178-191).-   3. Clarke described lipid content and composition of the Antarctic    krill Euphausia superba. Female total lipid content increases during    the summer as the ovary matures, and there is also some evidence of    an increase in the lipid content of males and immatures as winter    approaches. The storage lipid is mainly triacylglycerol and there is    less than 1% wax ester. Fatty acids are moderately unsaturated,    though less so in the ovarian lipid, and the triacylglycerol    contains up to 4% phytanic acid (Clarke A. 1984. Lipid content and    composition of Antarctic krill, Euphausia superba Dana. J. Crust.    Biol. 4:285-294).    -   Phleger et al. described lipid compositions of Antarctic        euphausiids, Euphausia superba, E. tricantha, E. frigida and        Thysanoessa macrura collected near Elephant Island during 1997        and 1998. Total lipid was highest in E. superba small juveniles        (16 mg g-1 wet mass), ranging from 12 to 15 mg in other        euphausiids. Polar lipid (56-81% of total lipid) and        triacylglycerol (12-38%) were the major lipids with wax esters        (6%) only present in E. tricantha. Cholesterol was the major        sterol (80-100% of total sterols) with desmosterol second in        abundance (1-18%). 1997 T. macrura and E. superba contained a        more diverse sterol profile, including 24-nordehydrocholesterol        (0.1-1.7%), trans-dehydrocholesterol (1.1-1.5%), brassicasterol        (0.5-1.7%), 24-methylenecholesterol (0.1-0.4%) and two stanols        (0.1-0.2%). Monounsaturated fatty acids included primarily        18:1(n-9)c (7-21%), 18:1(n-7)c (3-13%) and 16:1(n-7)c (2-7%).        The main saturated fatty acids in krill were 16:0 (18-29%), 14:0        (2-15%) and 18:0 (1-13%). Highest eicosapentaenoic acid [EPA,        20:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-3)] occurred        in E. superba (EPA, 15-21%; DHA, 9-14%), and were less abundant        in other krill. Lower levels of 18:4(n-3) in E. tricantha, E.        frigida and T. macrura (0.4-0.7% of total fatty acids) are more        consistent with a carnivorous or omnivorous diet as compared        with herbivorous E. superba (3.7-9.4%). The polyunsaturated        fatty acid (PUFA) 18:5(n-3) and the very-long chain (VLC-PUFA),        C(26) and C(28) PUFA, were not present in 1997 samples, but were        detected at low levels in most 1998 euphausiids        (Phleger, C. F. 2002. Interannual and between species comparison        of the lipids, fatty acids and sterols of Antarctic krill from        the US AMLR Elephant Island survey area. Comp Biochem Physiol B        Biochem Mol Biol. 131:733-747).-   4. Kolakowska described lipid compositions of seven krill (Euphausia    superba D.) samples, fresh and after various periods of storage at    251 K (Kelvin Degrees). Fresh krill lipid composition differed from    that determined in frozen samples, depending on storage duration,    season of harvest, and developmental stage. Phospholipids proved    most susceptible to changes, as opposed to triglycerides, which were    most resistant; diglycerides and cholesterol esters were also    destroyed. The freezing process per-se affected the lipid    composition only slightly; however, after 30 days storage the amount    of free fatty acids almost doubled. After 6 months storage at 251°    K, 70% of phospholipids were decomposed and the amount of free fatty    acids increased by a factor of 6 to 20. Monoglycerides, absent from    fresh krill, appeared after several months of frozen storage.    Juvenile krill were more susceptible to lipolytic changes. Females    bearing mature eggs contained stable phospholipids; only    triglycerides were hydrolysed (Kolakowska A. 1986. Lipid composition    of fresh and frozen-stored krill. Z Lebensm Unters Forsch.    182:475-478. Bottino described lipid compositions of two Antarctic    euphausiids. In Euphausia superba complex lipids were the major    lipid class followed by triglycerides. In E. crystallorophias the    complex lipids were also the major lipid class, but the second major    constituent was wax. The complex lipids of both Euphausiids    consisted mostly of phosphatidylcholine with smaller amounts of    phosphatidylethanolamine and lysophosphatidylcholine. The    phospholipids of E. crystallorophias were less unsaturated than    those of E. superba. The waxes of E. crystallorophias were mostly    esters of oleic (84%) and palmitoleic (10%) acids with    n-tetradecanol (69%) and n-hexadecanol (28%) (Bottino, N. R. 1975.    Lipid composition of two species of Antarctic krill: Euphausia    superba and E. crystallorophias. Comp Biochem Physiol B.    50:479-484).-   5. EP1997498 and WO02/102394 owned by Neptune Technologies &    Bioressources Inc. Relate to Neptune krill oil that corresponds to    acetone extracted krill lipids. Proteins and krill material are    removed from the lipid extract through filtration. The acetone and    residual water are removed by evaporation. The phospholipids content    is 38-50%, and EPA and DHA is 22-35%, wherein the major amount of    these omega-3's are attached to phospholipids.-   6. US2008/0274203 and WO 2008/117062 owned by Aker Biomarine ASA.    These applications disclose a new krill oil composition    characterized by high amounts of phospholipids, astaxanthin esters    and/or omega-3. This krill oil is characterized by comprising about    30-60% w/w phospholipids and about 20-35% omega-3 fatty acid wherein    the major amount of these omega-3 lipids are attached to    phospholipids.

The high content of polyunsaturated fatty acids in the phospholipidicfraction could be necessary to keep the plasmatic membrane fluidity atlow temperatures in the Antarctic oceans. A high unsaturation levelmight be required to give the krill phospholipids deposits the necessaryplasticity for the animal flectation and motion at low temperatures.

An increase of the total lipids present in the krill is accompanied withboth a decrease in the phospholipids and an increase in thetriglycerides. The polyunsaturated fatty acid content decreases as thecontent of total lipids increase (Saether et al. 1985. Loc. cit.).Post-mortem changes that occur in krill lipids showed that during thekrill storage at 0° C. the polyunsaturated fatty acids (20:5, 22:6)compared to the content of fatty acids (16:0) do not decrease. Thesedata suggest that during the krill storage at 0° C. a large oxidation ofpolyunsaturated fatty acids after the crustacean death does not occur.

There are several documents describing industrial processes to obtainkrill oil. These documents include:

-   1. Budzinski et al. (1986. Loc. cit.) and Saether et al. (1985. Loc.    cit.), described procedures for krill lipid extraction with    different organic solvents.-   2. CA2346979, ES2306527 or UA75029, documents presented by    Universite de Sherbrooke. This document described a method to    extract lipid fraction from marine and aquatic animals, including    krill, using acetone extraction.-   3. WO2006/106325 presented by Pro-Bio Group AS. This document    described a process to obtain phospholipids from krill. This process    comprises contacting the krill meal with an organic solvent to    produce a lipid containing liquid. Optionally this liquid is    extracted with other organic solvents to extract neutral lipids. The    remaining fraction is a phospholipids enriched fraction.-   4. EP1997498 and WO02/102394 presented by Neptune Technologies &    Bioressources Inc. These documents describe lipid extraction from    krill or krill derived material utilizing ketone solvents,    preferably acetone.-   5. GB407729 presented by Johan Olsen Nygaard. This patent describes    a method to extract oil from marine animals, in particular whale and    other sea mammals, by heating in bath of oil. This document is    different from our invention because is not applied to krill and    utilizes heated oil for lipid extraction. Our invention does not use    heated oil for phospholipids extraction.-   6. WO2009/027692 discloses a multiple step process for obtaining    krill meal and related products. The process includes a first    heating step in which krill is brought to a temperature of about    75° C. and thereafter sieved to obtain phospholipid free krill. This    phospholipid free krill is then heated a second time to 85° C.,    sieved again and pressed. The krill liquid (milk) left behind from    the first heating step is then coagulated to remove the proteins and    phospholipids.

These five documents describe the utilization of organic solvents,particularly acetone, to extract krill lipid fractions. These methodsbeing different from the procedure disclosed herein, as the methoddeclared in the present invention does not utilize organic solvents forextracting or purifying lipid fractions from Antarctic krill.

-   1. JP58008037, Nippon Suisan Kaisha Ltd. This document describes a    method to obtain eicosapentaenoic acid or derivatives from Antarctic    krill oil. Krill oil is converted into free fatty acid or an ester    thereof by the conventional method, e.g. saponification or    alcoholysis, being the resultant product continuously distilled to    collect a main fraction of distillate containing 40 wt % or more    titled substance. The main fraction of distillate is then treated    with urea to remove low unsaturated fatty acids. This document    differs from the present invention through the use of a distillation    process for lipid purification.-   2. JP2004024060 (Nippon Suisan Kaisha Ltd). This document describes    a procedure for obtaining an astaxanthin enriched lipid fraction    from Antarctic krill. This document declares a different process    from the one declared in the present invention in that the instant    process aim is not the astaxanthin production.-   3. US2008/0274203 (Aker Biomarine ASA). This document describes a    procedure for obtaining krill oil from krill meal using    supercritical fluid extraction in a two-stage process. Stage 1    removes the neutral lipid by extracting with neat supercritical CO₂    or CO₂ plus approximately 5% of a co-solvent. Stage 2 extracts the    actual krill oils by using supercritical CO₂ in combination with    approximately 20% ethanol. This document differs from the present    invention as this procedure does not use supercritical fluid    extraction.    -   WO2007/080514 (Krill A/S and Alfa Laval Copenhagen A/S),        describes a method for extracting lipid fractions from krill,        wherein freshly captured krill is grinded to obtain a slurry,        which is gently heated to a temperature below 60° C., preferably        bellow 30° C., for less than 45 minutes, thereafter the liquid        splits into an aqueous phase and a krill oil phase from which a        krill oil extract is derived without the use of organic        solvents. This document reveals a process different from the one        declared in the instant invention as the present procedure does        not grind the captured krill before heating. This document is        also different since the heating temperature declared in the        instant process is >90° C. Another main difference regarding the        process declared in this document is that grinded krill used to        produce slurry before heating produces an emulsification that        impedes the phospholipids separation by centrifugation. In the        present invention a slurry is not directly or indirectly        produced. Further in WO2007/080514 is used ultrasound for krill        oil separation while the present invention does not use any type        of ultrasound technology. It also proposes a simple extraction        while the present invention is based in a double-extraction        principle.-   4. WO2007080515 (Aker Biomarine ASA) describes a process for    obtaining krill lipids by processing the krill at a temperature    below 60° C. with mechanical and physical disruption of the lipid    cell membrane to facilitate low temperature extraction. This process    takes place under inert gas atmosphere to prevent oxidation or    denaturation of fat and proteins. Intermediate processing tanks are    kept at a minimum level to reduce residence time; and the oil after    recovery is immediately frozen to stabilize it. This document    differs from the present invention since the instant procedure does    not grind the captured krill to facilitate lipid extraction, instead    a heating temperature of >90° C. is used; further the process of the    present invention does not use any type of gases or freezing    technologies.-   5. WO2008/060163 (Pronova Biopharma Norge AS), describes a procedure    for obtaining krill oil-using CO₂ at supercritical pressure    containing ethanol, methanol, propanol or isopropanol. This document    is different from the present invention because the present    procedure does not use CO₂ supercritical lipid extraction with or    without solvent.-   6. WO2009/027692 (Aker Biomarine ASA), describes a method for krill    meal production. This procedure uses a two-step cooking process. In    the first step the proteins and phospholipids are removed from the    krill and precipitated as a coagulum. In the second stage the krill    without phospholipids are cooked. Following this, residual fat and    astaxanthin are removed from the krill using mechanical separation    methods. In this method, krill is heated to 60-75° C. in the    presence of water to dissolve/disperse lipids and proteins to the    water phase, called krill milk. This krill milk was heated to    95-100° C. to remove as a precipitate the krill protein and lipids    from the water phase. The processes of the present invention differ    from those disclosed in this document since the krill oil is not    separated from the crustacean by precipitation and the multiple    heating steps are avoided.

From current traditional krill meal processing on board, in some factoryvessels, only a small amount of krill oil is produced. This krill oil isusually enriched in neutral lipids with very low or undetectable amountof phospholipids (<0.5%). Normally, during the traditional on boardkrill process, fresh krill is heated using an indirect heating cookerwith rotating screw conveyor, followed by a twin-screw press and drier.The press liquid obtained by the twin-screw press is passed through adecanter to remove the insoluble solids. The clarified decanter liquidis then used to feed separators centrifuges to separate the krill oilnormally enriched with neutral lipids and astaxanthin. In thistraditional process the phospholipids are bound to the proteins in thepress cake. Therefore, phospholipids are usually found associated to thehill meal. In the krill fatty period, the fat content in the krill mealis usually 16-18%. In the same krill fatty period, the yield of theneutral lipid-enriched krill oil obtained using the traditional krillmeal plant is low, ranging usually between 0.3-1.0% from raw krill. Thisneutral lipid-enriched krill oil contains astaxanthin ranging between700-1.500 mg/kg depending on the season and the fishing ground catching.

Moreover, when a non-traditional krill meal processing layout is used, asimilar situation explained above was obtained. Normally, thenon-traditional krill meal plant considered a contherm cooker system, atwo-phase decanter or three-phase decanter and a drier. These decantersare used for de-watering and de-fatting the cooked krill. The decanterliquid is used to feed the centrifuge separators to obtain usually aneutral lipid-enriched krill oil with low or undetectable levels ofphospholipids (<0.5%). In this case, the phospholipids are also bound tothe proteins in the decanter solids. As described above, phospholipidsare found in the krill meal.

In this case, the yield of neutral lipid-enriched krill oil from thenon-traditional krill meal plant is much lower, in the fatty periodranging from 0.1 to 0.4% of raw krill. In this process a conventionalcontherm cooker system is used which has inherent agitation (scrapedknife). Therefore, the processed krill is agitated and also minced. Thisagitation/mincing process produce lipid emulsification along with krillproteins and water. Besides, krill phospholipids catalyze theemulsification because these lipids act as an emulsifier agent. For thisreason, using a non-traditional krill meal process, higher lipid contentis bound to the decanter solids. In the krill fatty period, the fatcontent of the decanter liquid has a lipid content ranging from 1.0 to2.5%, the resultant stickwater has a yellow color and it is emulsified,with a fat content ranging from 0.7 to 1.6%, the krill meal has a fatcontent ranging from 20 to 26%, and the krill oil recovery by thisnon-traditional process is very low.

The lipid composition and fatty acid profile of neutral lipid-enrichedkrill oil obtained using the traditional and non-traditional krill mealprocessing are very similar: triglycerides about 86-89%, phospholipidsnot detected (<0.5%), and DHA and EPA about 4-6%.

Several efforts have been made to produce a krill meal with a lower fatcontent and phospholipids enriched krill oil containing DHA and EPA,through an industrial scale method associated to a krill meal plant.Several different cooking temperatures, different decanting torque,strong pressing, using two decanting steps, washing the first decantersolids with stick water before the second decanter, electroplasmolysisand so on have been tested. However, the results have not beensuccessful.

Focusing on the problem to separate phospholipids-enriched krill oilwith DHA and EPA, some extraction methods have been developed and patentprotected. The patent application CL 1021 1995 (Compañía Tepual S.A)refers to a method for obtaining krill oil using thermal fractionationand centrifugation. This oil obtaining process claims that the fattyacids types and lipids composition can be regulated controlling thekrill cooking temperature during the process of oil production. At highcooking temperature (95° C.) lower yield of poly-unsaturated andphospholipids fractions were obtained as compared when the cookingtemperature was reduced (75° C.). Also this oil process claims that maincomponents of its fractional composition correspond to triglyceridesbeing 35 to 96%; and phospholipids from 4 to 28%. The poly-unsaturatedfatty acids ranged from 4 to 46%. Basically this process used forobtaining krill oil, considers krill cooking, pressing the cooked krilland the passing the press liquid through a two-phase decanter toseparate the insoluble solids, being the oil separated from the decanterliquid using centrifuge separator. During this process only one type ofkrill oil was obtained.

SUMMARY OF THE INVENTION

This invention discloses a new on board (at sea) and/or on shore (onland) process for simultaneously obtaining both: 1) phospholipidsenriched krill oil and 2) neutral lipid enriched krill oil containingDHA and EPA poly-unsaturated fatty acids and astaxanthin. Antarctickrill oil is particularly rich in DHA and EPA containing phospholipids.Because the amphipathic nature of phospholipids, i.e. a negative chargedphosphate group in one end and a hydrophobic lipid in the other end,this lipid is a very potent emulsifier agent. The grinding and/ormincing of krill induces emulsification of soluble proteins, water,neutral lipids and phospholipids. Therefore, once the emulsion isformed, phospholipids can be obtained from krill mixtures only byorganic solvent or CO₂ supercritical extractions. See WO02/102394 andUS2008/0274203 as example. The present invention advantageously avoidsthe use of these extractors and, as mentioned below, the grinding and/ormincing of the krill during the process.

In the present invention, a new procedure for phospholipids extractionfrom fresh krill or other similar crustaceans is disclosed. Thisprocedure is based on a process where no grinding, agitation and/ormincing of the krill is carried out. Using this method, no orsubstantially no phospholipid emulsification occurs. Cooking of wholeintact krill or fractions produces a first solid fraction and a firstliquid fraction, with the first liquid fraction being released from thecrustaceans. The oil contained in this liquid phase (i.e. the firstliquid fraction) is enriched with neutral lipids. The first solidfraction obtained from cooking can be further processed to obtain asecond liquid fraction and a second solid fraction. The oil obtainedfrom this second liquid fraction is also enriched with phospholipids.The second solid fraction is processed to obtain a low fat krill meal.

The present invention allows a much more efficient extraction processand is preferably done almost immediately after raw or fraction of krillhas been harvested at sea. The present invention is preferably doneonboard, which allows having a much more controlled extraction process,allowing a dramatic reduction in not-necessary logistic transshipment,raw material and cargo handling. Other advantages of the presentinvention include the “finished product” character of the end productwhich is obtained on the same premises where capture and processingtakes place.

The process of the present invention can also be used with other similarcrustacean species, whether they are farmed or captured crustaceans,such as but not limited to Pandalus borealis, Cervimunida johni,Heterocarpus reedi, Pleuroncodes monodom, Penaeus vannamei, Penaeusmonodon, Penaeus stylirostris, Penaeus chinensis, Penaeus. orientalis,Penaeus japonicus, Penaeus indicus, Penaeus merguiensis, Penaeusesculentus, Penaeus setiferus, Macrobrachium spp, and others. Moreover,the processes described herein can be used to obtain oil, extracts anduseful compositions from various fish and/or algae species. In suchembodiments, the processes are carried out but the krill is replacedwith either fish or algae.

In accordance with one aspect of the invention, a solvent-free processfor producing krill oil is provided which includes:

-   -   a) cooking krill, which is preferably fresh, for a time and at a        temperature sufficient to transform the krill into a form        capable of being separated into a first solid fraction and a        first liquid fraction while substantially avoiding        emulsification of the krill;    -   b) separating the first solid fraction and the first liquid        fraction from the cooked krill; and    -   c) obtaining krill oil from either the first liquid fraction or        the first solid fraction, said separating and said obtaining        steps being carried out without the use of solvents.

In a related aspect of the invention there is provided a solvent-freeprocess for producing krill oil which includes:

a) cooking krill, which is preferably fresh, in a vessel for a timesufficient to cause the krill reach a temperature of at least about 90°C. and transforming the krill into a form capable of being separatedinto a first solid fraction and a first liquid fraction whilesubstantially avoiding emulsification of the krill;b) removing the cooked, transformed krill from the vessel at saidtemperature of at least 90 degrees;c) separating the first solid fraction and the first liquid fractionfrom the cooked, transformed krill; andd) obtaining the krill oil from either the first liquid fraction or thefirst solid fraction, said process being carried out without the use ofsolvents.

To obtain the krill oil from the first liquid fraction, the first liquidfraction is separated into krill oil and water.

As noted above, the first solid fraction which results from cooking is,preferably, further processed by:

-   -   (a) squeezing the first solid fraction to obtain a second solid        fraction and a second liquid fraction; and    -   (b) separating the second liquid fraction into a krill oil and        water.

Preferably, the first liquid fraction contains a krill oil enriched withneutral lipids and the second liquid fraction krill oil is preferablyenriched with phospholipids

Separating the second liquid fraction into krill oil and water issuitably accomplished by using a separator to separate the oil from thewater, or by drying to remove the water from the oil.

As a part of the present innovation, this invention is carried out withequipment that avoids or minimizes the phospholipids forming emulsionswhile separating the phospholipids from the krill. The equipment ischosen from conventional equipment and is operated to avoid agitation,mincing, and/or grinding, throughout all processing steps (e.g. cooking,pumping, and so on) to reduce oil emulsification with protein, water andkrill phospholipids, this last component acting as the emulsifier agent.Such equipment include screw pumps, continuous cookers with screwconveyor at low speed, belt conveyors, screw conveyors, de-boner,chopper or similar.

Other equipment possible to use, that avoids agitation and/or grinding,throughout all processing steps include:

1. Krill and other crustaceans peeling machines2. Krill and other crustaceans brush peeling machines3. Krill and other crustaceans disk peeling machines4. Krill and other crustaceans coagulator machines working at low speed5. Krill and other crustaceans dewatering machines working at low speed6. Krill and other crustaceans deboning machines.

This invention relates to a method for obtaining specific Krill oilsfrom Krill, preferably from South Antarctic Krill, through a processingpreferably made on board (at sea), on board factory trawlers, or mothervessels. Further the present invention is suitable for being carried outon shore (on land) processing plants. The final processing target of theprocessing method disclosed in this invention includes: a) cooking ofwhole or fraction fresh marine species without agitation and/or mincing;b) separating of cooked marine species using, for example, a two-phasedecanter and/or a three-phase decanter or any other type of separator toobtain a partial de-fatted and de-watered solid, a first solid fraction,and a decanter liquid, a first liquid fraction,) c) squeezing of thepartial de-fatted and de-watered decanter solid through a press and/orusing a twin-screw press and/or any other pressing device to obtain apress liquid, a second liquid fraction, and a solid fraction, a secondsolid fraction, which can be further processed to obtain the low fatdried marine-species meal; d) centrifuging of the press liquid to obtainthe phospholipids enriched marine-species oil; e) separating of thedecanter liquid obtained in step b) to obtain the neutral lipid enrichedmarine-species oil and stick water. The equipment used in this processavoids agitation throughout all steps (i.e. cooking, pumping, and so on)to reduce oil emulsification with protein, water and marine-speciesphospholipids, acting this last component as the emulsifier agent.

This invention also includes a phospholipids-, neutral lipids-,omega-3's- and protein-enriched dried powder, and the use of thisproduct as a heath product for human application, a powerfulhealth-promoter, growth enhancer, immune-system promoter and wellnessend-product. All products obtained with the procedure disclosed in thisinvention are produced by a non-chemical treatment, i.e. a process freeof organic solvents and/or CO₂ supercritical fluid, for separating thekrill oil.

The phospholipids enriched krill oil can be broadly defined asincluding:

-   -   (a) phospholipids in an amount of about 30 to about 70% by        weight;    -   (b) DHL and EPA in an amount of about 10 to about 70% by weight;    -   (c) neutral lipids in an amount of about 30 to about 70% by        weight;    -   (d) astaxanthin in an amount of about 200 to about 1,500 mg/kg;    -   (e) phosphatidylserine in amount of about 1.5% by weight or more        total lipids;    -   (f) lysophosphatidylcholine in an amount of about 0.6% or less        by weight total lipids; and    -   (g) free fatty acids in an amount of about 3.4% or less by        weight total lipids.

The Phosphatidylserine (PS) content of the PL (phospholipid)-enrichedkrill oil obtained as a result of the present invention, about 1.5% ormore, is believed to be unique as compared to that obtained using priorart processes, i.e. those using solvents and/or CO2 supercritical toextract PL-enriched krill oil. In those prior art processes, PS contentis either nil or less than 0.5%.

The phospholipids enriched krill oil of the present invention is usefulfor human health applications and has a total phospholipids content ofabout 30 to 70% w/w, preferably from about 35 to 60% w/w, and morepreferably from about 35 to 55% w/w. The DHA and EPA content are, incombination, from about 10 to 70% w/w, preferably from about 15 to 60%w/w, and more preferably from 20 to 55% w/w. Neutral lipids content isfrom 30 to 70% w/w, preferably from about 40% to 65% w/w, and morepreferably from about 45 to 65% w/w. Astaxanthin content is from about200 to 1,500 mg/kg, preferably from about 300 to 1,200 mg/kg, and morepreferably from about 400 to 1,000 mg/kg.

The neutral lipid enriched krill oil can be broadly defined asincluding:

-   -   (a) neutral lipids in an amount of about 50 to about 100% by        weight;    -   (b) DHA and EPA in an amount of about 2 to about 45% by weight;    -   (c) phospholipids in an amount of less than about 10% by weight;    -   (d) astaxanthin in an amount of about 200 to about 1,500 mg/kg;        and    -   (e) free fatty acid content in an amount of about 3.4% or less        by weight.

The neutral lipid enriched krill oil of the present invention is alsouseful for human health applications, having a content of neutral lipidsfrom 50 to 100% w/w, preferably from 60 to 100% w/w, and more preferablyfrom 70 to 100% w/w. DHA and EPA content are from 2 to 45% w/w,preferably from 2 to 40% w/w, and more preferably from 5 to 35% w/w.Phospholipids content is less than 10% w/w, preferably less than 5% w/w,and more preferably less than 2% w/w. Astaxanthin content is from 200 to1,500 mg/kg, preferably from 300 to 1,200 mg/kg, and more preferablyfrom 400 to 1,000 mg/kg.

The phospholipids-enriched krill oil of the present invention ispreferably produced on board (at sea) which provides having the directadvantage of using of a fresh raw krill, this way avoiding freezing ofthe whole or fraction raw of krill. In addition to the freshness of thesaid end material, on board processing also produces fresh krill meal,which can eventually be used for further krill oils extraction processesin on shore (on land) processing plants. In alternative aspects of theinvention, the process can be carried out on land using fresh or frozenkrill previously obtained from the sea, which would be preferably be atleast partially thawed before being cooked, or krill portions previouslyprocessed in some manner.

These preferably on board (at sea) krill oil processing methods haveadditional and important advantages such as avoiding frozen raw krilltransportation from at sea operations (on board) to on shore (on land)port facilities and further transportation to on shore (on land)processing plants, this way the end-products of present invention have asignificantly lower cost structure.

The phospholipids-enriched krill oil of the present invention, which ispreferably produced using on board (at sea) processes. That means theassurance of the use of highly fresh whole or fraction raw krill,avoiding phospholipids decomposition and/or lipids deterioration whichmay occur during certain freezing process, frozen transportation andfrozen storage typically required when prior art processes arecarried-out on land (on shore) premises.

This invention represents a potent krill-related environment-drivendevelopment, as among its direct benefits, it includes:

-   a) An overall lower catch-volume requirement to secure the same    amount output of end product as compared to other methods as there    is a more efficient processing which allows less captured-tonnage    per end-product-tonnage;-   b) a significant reduction in processing infrastructure in relation    to any other comparable processing method as the amount of catch and    equivalent processing capacity is diminished due to the onboard    processing;-   c) a relevant improvement of the end-product quality in terms of    freshness and its molecules quality, which means that the benefit to    the end-user, be it for human health or animal nutrition, will be    through the reduction of the market-cost per equivalent high-quality    molecule;-   d) this invention guarantees lower processing costs which goes to    the direct benefit of end-users through a lower end-product selling    price;-   e) the biomass protection is also guaranteed as the value-added    concept is secured on board (at sea) and does not need extra costs    to transport raw material for further on shore (on land) processing;-   f) this invention allows having end-products secured on board (at    sea);-   g) This invention will also provide a much better resource    protection as the catch effort will not be focused on a limited    catch and processing period rather spread-out the entire krill    fishing season.

This invention further relates to South Antarctic Krill processing madepreferably on board (at sea) factory trawlers or mother vessels. Thisinvention also relates to krill processing made on shore (on land)processing plants whereas the on board (at sea) processing stepsprevails on shore (on land). The final processing target of theprocessing method disclosed in this invention includes:

-   a) cooking of whole and fresh marine species without agitation and    or grinding;-   b) decanting of cooked marine species, i.e. krill using, for    example, a two-phase decanter and/or a three-phase decanter and/or    any other type of solid-liquid separator to obtain a partial    de-fatted and de-watered solid and a decanter liquid;-   c) squeezing of the partial de-fatted and de-watered decanter solid    through a press, such as, for example, a twin-screw press to obtain    a press liquid and a solid fraction which is further processed to    obtain the low fat dried marine-species meal;-   d) centrifuging of the press liquid to obtain the phospholipids    enriched marine-species oil; and-   e) centrifuging of the decanter liquid obtained in step b) to obtain    the neutral lipid enriched marine-species oil and stick water, i.e.    the water remaining after the oil is removed from the aqueous phase.    Stick water contains a substantial percent of the total protein as    well as other soluble components of the krill.

This process uses equipment that avoid or substantially avoid agitationthroughout all steps (i.e. cooking, pumping, and so on) to reduce oilemulsification with protein, water and marine-species phospholipids,which act as the emulsifier agent.

The on board (at sea) processing includes either on board factorytrawlers, mother vessels acting as processors only, mother vesselsacting as catchers and processors or any other combination for mothervessels, onboard tramper vessels used as processing factories and/or anyother at sea processing factory vessel and/or any other at seaprocessing factory layout operation for this specific purposes, beingthe resulting marine-oils obtained either as a by-product or as a finalproduct or a combination of both.

The krill meal obtained with the process of the present invention has afat content ranging from 5 to 15%, protein content from 60% to 70%, andmoisture content from 6 to 10%.

Because both krill oil compositions obtained in the present inventionare not exposed to the use of any toxic or non-toxic chemicals duringthe process, the resulting krill oils of this invention could be safelyused for human consumption according to the European Food SafetyAuthority (EFSA) (Scientific Opinion of the Panel on Dietetic ProductsNutrition and Allergies on a request from the European Commission of thesafety of “Lipid extract from Euphausia superba” as food ingredient. TheEFSA Journal (2009). 938, 1-16).

Human health applications for the krill oils obtained in the presentinvention, can be without limitation for reducing premenstrual symptoms,preventing hypertension, control of blood glucose levels in patients,control of arthritis symptoms, prevention of hyperlipidemia and otherhealth applications. Such oils can also be used in preparingdermatologic products, specially topic or systemic products, fortreating skin diseases related to a deficiency of essential fatty acids,such as xerotic skin, hyperkeratosis, ictiosis, acne, dermic ulcers,psoriasis, serborreic eczema, atopic dermatitis, among others.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 and 2 are flowcharts depicting the process of the presentinvention;

FIG. 3 shows a flowchart for a process to obtain neutral lipids richkrill oil in accordance with the invention; and

FIG. 4 shows a flowchart for a contherm process to obtain krill oilreviewed in Table 2 below.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it should beunderstood that the invention as described is not to be limited in itsapplication to the process details described herein. The invention assuch, embraces other embodiments and various ways for being applied. Itis also should be understood that the phraseology or terminology usedherein is for the purpose of description and it is not intended for anytype of limitation.

The following description may be more readily understood by reference toFIGS. 1 and 2.

The present invention provides systems and processes for processing amarine biomass. As a preferred embodiment, the marine biomass is krill,preferably the Antarctic krill Euphasia superba. Other krill species orcrustacean can also be processed using the systems and processes of thepresent invention. Examples of such species are E. crystallorophias, E.frigida, E. tricantha, E. vellantini, E. lougirostris, E. lucens, E.similis, E. spinifera, E. recurva, E. pacifica, Thysanoessa macrura, T.vicinia, T. gregaria, T. raschii, T. inermis, Pandalus borealis,Cervimunida johni, Heterocarpus reedi or Pleuroncodes monodom. The krillis preferably processed in a fresh state as defined herein. Moreover,the processes described herein can be used to obtain oil, extracts andother useful compositions from various fish and/or algae species. Inaccordance therewith, any of the processes described herein is modifiedto replace the krill with the desired fish and/or algae species.

As a preferred embodiment, the krill is harvested 10, see FIG. 1, at seain a conventional manner using conventional equipment and then processedfresh on board a fishing vessel either factory trawler, mother factoryvessels, or intermediate processor or similar or whatever other shipsuitable to carry out the process of this invention within a term of 14,12, 10, 8, 6, 5, 4, 3, or preferably 2 or 4 hours of catching krill. Insome embodiments, the krill is processed on board a ship within 1 houror preferably 0.5 hours or more preferably within 20 minutes aftercatching the krill. Within the embodiments of the present invention, itis included that the ship tows a trawl that is configured to catch thekrill and/or that the ship receives its krill or other species fromfishing vessels or other factory trawlers. The krill is then transferredfrom the trawl to the ship and processed, preferably immediately afterkrill catching. The trawl comprises (either a regular fishing gearcomposed of trawls and/or purse seining system and/or) a pumping systemto pump the freshly caught krill from the trawl to the ship so the krillcan be processed in a fresh state. In some embodiments the catch system,be it regular trawl or pump system, it is designed in such a manner thatsubstantially no damage, (deterioration) grinding or mincing is done tothe krill during the pumping from the trawl to the ship.

In a preferred embodiment, the fresh whole krill or fraction istransported to the cooker or cooker vessel 12 using belt conveyor, orscrew conveyor or screw pumping or another transport system avoidingkrill agitation to avoid the oil emulsification with protein and water.Sea water drains from the krill and krill is preferably washed withfresh water prior to feeding the cooker.

The whole krill is cooked, i.e. heated, in a cooker vessel with indirectand/or direct steam heating or another heating system. Combinations ofindirect heating and live steam injection can also be used. Cooking(hereinafter used so as to include heating) can be a batch or continuousprocess. Preferably, continuously using a rotating cooker which injectssteam directly into the krill (or flow containing the krill). Suitably,a horizontal cooker with an internal rotating screw is used. The screwrotates at a low rotation speed of about 1-100 rpm, preferably 2-20 rpm,and more preferably 5-10 rpm, without agitation to avoid the oilemulsification. The krill temperature at entrance to the cooker is atatmospheric temperature and the temperature of the krill at the exit ofthe cooker is preferably at least about 90° C., more preferably about90-100° C. and, in some embodiments even more preferably about 93-95°C., or whatever other temperatures not disclosed here but that arenecessary to reach complete protein denaturation. The heating of thekrill also allows the krill-containing flow, i.e. cooked krill, to berendered into two phases for later separation: a first solid krillfraction and a first liquid krill fraction. The cooker type is notparticularly restricted but as preferred embodiment this cooker mustoperate without agitation for avoiding the emulsification state. Theamount of time the krill is cooked will depend upon several factorsincluding but not limited, the type of cooker used, i.e. batch orcontinuous, size and temperature, rate of throughout. In someembodiments, the cooking time for batches will be from about 10 to about20 minutes, while cooking times for continuous processes can havesimilar time ranges, which corresponds to the approximate residence timeof the krill in the continuous cooking device without causingsignificant emulsification of the krill phospholipids.

In some embodiments, the cooked krill is transported to a separator ordecanter or separating decanter 14, preferably a two-phase decanter,using a screw conveyor or a screw pumping, or another transportingsystem without krill agitation for avoiding oil emulsification. Aseparator is used to separate the cooked krill into a first solidfraction and a first liquid fraction. Any conventional liquid-solidseparator can be used. Suitably, centrifugation is used to acceleratethe separation of solid and liquid. Suitably, a centrifuge separatorsuch as decanter centrifuge is used. In some preferred embodiments, adecanter separator is used for separating the solid and liquid. Inalternative embodiments, alternative separators such a horizontaldecanter separators or similar apparatus can be used.

The cooked krill is preferably passed through a two phase-decanter withhigh torque about 1-10 kNn, preferably 1.2-5 kNn, more preferably 1.5-3kNm, even more preferably 1.8-2.5 kNm, and a speed of about 100-10,000rpm, in some embodiments, preferably 1,000-8,000 rpm, more preferably2,000-5,000 rpm, even more preferably 3,000-4,600 rpm, for partialde-watering and partial de-fatting, separating it into a decanter solidsphase and a decanter liquid phase. In some alternative embodiments, thecooked krill separating is conducted using a decanter operated at speedabout 3.000 rpm or higher. The decanter type is not particularlyrestricted. The moisture content of decanter solid is about 40-80%,preferably, 50-70%, more preferably 55-67%, even more preferably 58-65%.

Table No 1 shows the lipid and moisture content of decanter solids anddecanter liquids, (corresponding to the first solid and first liquidkrill fractions, respectively) obtained with a krill catch during fattyperiod using a cooker with indirect and direct steam heating with ascrew conveyor at low rotation speed (i.e. about 2-10 rpm). The decantersolid has a lipid content range of 19.8 to 22.4% wt dry base, so theresulting krill meal with 8% of moisture from this solid will have alipid content of about 18.2% to 20.6%. These results show that the useof a two-phase decanter alone is not enough to achieve a good de-fattingof the cooked krill to obtain more krill oil and krill meal with a fatcontent lower than 18%. Anyway, the decanter solids obtained from acooker operating a at a low speed have a lower lipid content and lowernon-emulsified lipids than that of decanter solids obtained usingprocesses in which a contherm cooker is used. The processes of thepresent invention which involve using a cooker working at a lower speedof rotation advantageously make it possible to avoid emulsification.Moreover, the inventive processes allow the resulting lipids be foundmore in the liquid fraction instead of the solid fraction. Conversely,when a contherm type cooker which works at a higher speed of rotation(above 500 rpm), emulsification is generated and, as seen in Table 2,below, lipids are retained more in the solid fraction and less in theliquid fraction.

TABLE 1 Lipid and moisture content of decanter solids and decanterliquids obtained with krill captured during South Antarctic's Krillfatty period using a cooker with indirect and direct steam heatingsystem with screw conveyor operating at a low rotation speed. See alsoFIG. 2 Flow Chart. De- Krill canter Tem- Moist- Total Lipids LipidsKrill Torque perature ure Solids WB DB Material (kNm) (° C.) (%) (%) (%)(%) Decanter 2.0 94 63.1 36.9 7.3 19.8 Solids Decanter 93.2 6.8 1.6 23.5Liquid Decanter 2.1 93 62.9 37.1 8.1 21.8 Solids Decanter 92.7 7.3 1.824.7 Liquid Decanter 2.0 94 61.6 38.4 7.8 20.3 Solids Decanter 91.3 8.71.9 21.8 Liquid Decanter 2.0 93 63.0 37.0 8.3 22.4 Solids Decanter 91.18.9 1.9 21.3 Liquid WB: wet base DB: dry base

In some embodiments of the present invention, using a cooker calibratedfor a low rotation speed of the screw conveyor in a continuous processof preferably about 2-10 rpm. The decanter liquid obtained in suchcookers is not emulsified and has higher lipid content compared to thedecanter liquid obtained using a conventional contherm cooker.

Following the flow chart of FIG. 4, Table No 2 shows the lipid andmoisture content of decanter solids and decanter liquid, wherein acontherm cooker has been used. This equipment is not preferred withinthe embodiments of this invention, as a large proportion of lipidsremain in the decanter solids, and the lipids contained in the decanterliquid are emulsified. Krill oil recovery using this equipment was verylow and the resulting krill meal has a high fat content, specificallyover 20% when obtained in the krill fatty period.

TABLE 2 Lipid and moisture content of decanter solids and decanterliquids obtained with krill captured during South Antarctic's Krillfatty period using a contherm cooker system See FIG. 4 Decanter KrillTotal Lipids Torque Temperature Moisture Solids Lipids DB Krill Material(kNm) (° C.) (%) (%) WB (%) (%) Decanter 2.1 95 63.1 36.9 8.7 23.6Solids Decanter 92.7 7.3 1.5 20.5 Liquid Decanter 2.0 96 61.9 38.1 9.324.4 Solids Decanter 92.1 7.9 1.7 21.5 Liquid Decanter 2.0 94 63.0 37.08.4 22.7 Solids Decanter 92.3 7.7 1.5 19.5 Liquid Decanter 2.0 95 63.636.4 8.6 23.6 Solids Decanter 92.8 7.2 1.7 23.6 Liquid WB: wet base DB:dry base

While applicants are not bound by theory, it is believed that thecombination of the preferred decanting and pressing conditions describedherein result in obtaining krill oil compositions having higher levelstriglycerides and phospholipids while minimizing emulsion formation byvirtue of limited rotational speeds for the device during cooking Suchdesirable results are not available when contherm cooking apparatus withtheir higher rotational speeds are employed. Thus, the low rotationalspeeds mentioned herein, i.e. about 1-100 rpm, preferably 2-20 rpm, andmore preferably 5-10 rpm, are used, without agitation of the krill beingcooked, to avoid the oil emulsification.

Turning now to the flow chart of FIG. 2, some details are providedregarding a preferred embodiment in the process of the presentinvention. After cooking and decanting of the krill, the obtaineddecanter liquid with neutral lipids and astaxanthin is passed through acentrifuge separator to separate the first liquid fraction (decanterliquid) into oil and water. Any conventional liquid-liquid separator canbe used. Suitably, centrifugation is used to accelerate separating theliquids. Suitably, a centrifuge separator is used. After the separatorcentrifuge a neutral-enriched krill oil and a first stick water (stickwater 1 in flow chart) is obtained. The oily fraction is passed againthrough a purifier separator centrifuge (identified in the flow chart asPolish Centrifuge #1), to separate the krill oil enriched with neutrallipids with astaxanthin and substantially free or without (or nodetectable) phospholipids from additional stick water (not shown in theflow chart). The obtained stick water (Stick water 1 in the flow chart)has a low fat content, with a maximum of about 0.3-0.5%, in anon-emulsified form suitable for further processing (concentrateproduction) and sludge. Stick water can be further evaporated to form aconcentrate. See also FIG. 3 for a related process for obtaining krilloil, press cakes, etc.

In some embodiments, there is provided a process for obtaining theneutral lipid-enriched oil which, has an exclusive and separated lineincluding: separator centrifuge, purifier separator centrifuge, pumps,piping, heat exchangers, tanks and packaging station. As such, there isprovided an organic solvent-free process for obtaining krilloil-containing compositions having the steps of a) cooking krill in avessel for a time and at a temperature sufficient to denature theprotein content of the krill and cause a first solid krill fractionbeing partially defatted and de-watered and a first liquid krillfraction to be formed while substantially avoiding emulsification of thefirst solid and first liquid krill fractions; b) removing the firstsolid and first liquid krill fractions from the cooker vessel at atemperature of at least about 90° C.; c) separating the first solidfraction and the first liquid fraction; and d) centrifuging the firstliquid portion obtained as a result of step b) to obtain an organicsolvent free krill oil composition enriched in neutral lipids and stickwater and optionally removing the stick water therefrom.

In a preferred embodiment of the process of the present invention, thedecanter solids phase or, the first solid krill fraction, is fed to apress 26 using a screw pump or a screw conveyor or other feeding systemwherein agitation does not occur. A press is used to express the secondliquid krill fraction 28 from the first solid krill fraction and form asecond solid fraction 29. Such presses are conventional and include, forexample, single press, twin screw press, double or other presses know tothose of ordinary skill. Suitably, squeezing is conducted preferably ina continuous process but may be a batch process. Suitable pressesinclude box presses, platen presses, pot presses, cage press, for abatch process and screw presses, and roller presses for a continuousprocess. In one aspect of this embodiment, a two screw press is usedwhich squeezes the first solid fraction and separates the squeezed firstsolid fraction into the second solid fraction and a second liquidfraction. The decanter solids phase is pressed using twin screw presswith high pressing force or using another pressing system to release theoil with phospholipids 36 linked to the denatured protein according tothe production line for the product of the present invention. As apreferred embodiment, the pressing step is carried out by continuouspressing at a full feeding condition using a 2-10 rpm speed, and morepreferably 3-6 rpm and a decanter solids temperature feeding of 90-96°C. and more preferably 93-95° C. The pressing system is not particularlyrestricted. As described above, the decanter solids phase keeps all ofthe phospholipids inside of the coagulated protein, then a strongpressing of the decanter solids releases an important high percentage,i.e. up to about 30 to 60%, of the phospholipids to the press liquid.The moisture of this press cake is about 45-55%, preferably 48-53%.Table 3 shows the lipid and moisture content from the press cake of thepresent invention at different feeding levels of the twin press in thekrill fatty period.

TABLE 3 Moisture Dry Solids Lipids WB Lipids DB (%) (%) (%) (%) PressCake 1 58.1 41.9 8.6 20.5 Press Cake 2 58.6 41.4 8.4 20.3 Press Cake 356.9 43.1 8.0 18.5 Press Cake 4 56.7 43.3 8.0 18.5 Press Cake 5 54.645.4 7.4 16.3 Press Cake 6 54.5 45.5 7.2 15.8 Press Cake 7 54.3 45.7 5.512.0 Press Cake 8 53.9 46.1 5.6 12.1 WB: wet base DB: dry base

Comments

Press Cake samples 1 to 6 were taken when the press was not fully fedwith decanter solids

Press Cake samples 7 & 8 were taken when the press was fully fed withdecanter solids

Table No 4 shows this press liquid, second liquid fraction, 28,composition in the krill fatty period. According to the process orsystem disclosed in the present invention, this press liquid has a highfat content in the range of about 3-25%, preferably 5-20% and morepreferably 8-17% (wet base), depending on the seasonal lipid content ofkrill and if it is not in an emulsified form. In the traditional krillmeal processing using only a twin-screw press (without prior use of adecanter), the fat content in this press liquid is lower than 0.5-3%(wet base) depending on the lipid content of krill and if it is not inan emulsified form.

TABLE 4 Press Liquid Press Liquid Press Liquid Press Liquid Composition1 2 3 4 Moisture (%) 78.3 77.7 77.3 80.4 Proteins (%) 5.8 6.1 6.2 5.2Lipids (%) 16.9 14.0 15.3 13.2

In some embodiments of the present invention, there is also provided adried complex with oil containing phospholipids with DHA and EPA,proteins and astaxanthin obtained by drying 30 the press liquid “as-is”without centrifuge separation. Such dried complex 32 corresponds to ahuman-grade krill-related product for being used in many human healthapplications. See also FIG. 2 in this regard. Drying is done in aconventional manner using conventional equipment. In accordancetherewith a process is an organic solvent-free process for obtainingkrill oil-containing compositions, having the steps of a) cooking krillin a vessel for a time and at a temperature sufficient to denature theprotein content of the krill and cause a first solid krill fractionbeing partially defatted and de-watered and a first liquid krillfraction to be formed while substantially avoiding emulsification of thefirst solid and first liquid krill fractions; b) removing the firstsolid and first liquid krill fractions from the cooker vessel at atemperature of at least about 90° C.; c) separating the first solidfraction and the first liquid fraction; d) squeezing the separated firstsolid fraction and obtaining a press liquid and a second solid portiontherefrom; and e) centrifuging and drying the press liquid obtained as aresult of step c) to obtain an organic solvent free dried complexcontaining krill oil in combination with phospholipids, DHA, EPA,proteins, astaxanthin and neutral lipids.

In some embodiments, a synergic action of the two-phase decanter andfurther pressing with twin-screw press or another strong pressing systemis used to release the phospholipids with DHA and EPA in the oil, mixedwith astaxanthin and neutral lipids. The press liquid, at a temperatureof about 25-121° C., preferably 50-110° C., more preferably 80-100° C.and even more preferably 90-96° C., is pumped to the separatorcentrifuge 34, using a screw pump or other feeding system, avoidingagitation, wherein the krill oil with phospholipids with DHA and EPAmixed with astaxanthin and neutral lipids 36 is separated.

The separator that is used to separate the second liquid into oil andwater is any conventional liquid-liquid separator. Suitably,centrifugation is used to accelerate the separator of the liquids.Suitably, centrifugation is used to accelerate the separator of theliquids. Suitably, a separator centrifuge is used. The processing speedin the centrifuge separator operates at 4,000-8,000 rpm, more preferablyat 4,600-6,800 rpm, with an automatic periodic discharge of solids. Inone embodiment of the present invention, the oil separator centrifuge isnot particularly restricted, i.e. any centrifuge equipment satisfyingthe indicated conditions is suitable for being used. This krill oil isonce more centrifuged through a purifier separator to clarify it,operating at a processing speed of 5,000-10,000 rpm, more preferably at6,000-8,200 rpm, with an automatic periodic discharge of solids forlater packages. In the embodiments of the present invention, thepurifier separator centrifuge is not particularly restricted, i.e. anycentrifuge equipment satisfying the indicated conditions is suitable forbeing used. The stickwater 38 from separator 34 is fed to evaporator 22to form concentrate 24.

As an additional preferred embodiment, the krill oil with phospholipidswith DHA and EPA mixed with astaxanthin and neutral lipids, product ofthe present invention, is obtained using an exclusive and separated oilprocess line including: a separator centrifuge, a purifier separator,pumps, piping, heat exchangers, tanks and a packaging station,completely different and separated from the other oil line with neutrallipid from the decanter liquid phase.

Examples 3 and 4, infra, show the characteristics of krill oils obtainedin the present invention:

I). the neutral lipid enriched krill oil of the present invention isalso useful for health human application having a content of:

-   -   neutral lipids from 50 to 100% w/w, preferably from 60 to 100%        w/w, and more-preferably from 70 to 100% w/w,    -   DHA and EPA content are from 2 to 45% w/w, preferably from 2 to        40% w/w, and more preferably from 5 to 35% w/w,    -   Phospholipids content is lower than 10% w/w, more preferably        lower than 5% w/w, and more preferably lower than 2% w/w,    -   Astaxanthin content is from 200 to 1.500 mg/kg, preferably from        300 to 1200 mg/kg, and more preferably from 400 to 1,000 mg/kg.

II) the phospholipids enriched krill oil of the present invention can beuseful for health human applications, having a content of:

-   -   total phospholipids content from 30 to 70% w/w, preferably from        35 to 60% w/w, and more preferably from 35 to 55% w/w,    -   DHA and EPA content is from 10 to 70% w/w, preferably from 15 to        60% w/w, and more preferably from 20 to 55% w/w,    -   Neutral lipids content is from 30 to 70% w/w, preferably from        40% to 65% w/w, and more preferably from 45 to 65% w/w,    -   Astaxanthin content is from 200 to 1.500 mg/kg, preferably from        300 to 1.200 mg/kg, and more preferably from 400 to 1.000 mg/kg.

According to the above declared compositions for both oil products,obtained through the process of the present invention, these oils aresuitable for health human applications.

From the above, considering the composition characteristics of thiskrill oil and the process for obtaining the same, it can be concludedthat the process of the present invention provides a krill oil productcontaining DHA and EPA in the phospholipids fraction with astaxanthinand neutral lipids, which is produced through a different process, whichis new and improved, regarding all those krill oils obtained throughprocesses involving solvent extraction and/or using supercritical fluidextraction or through thermal fractionation and centrifugation;additionally, the resulting krill meal, in any Antarctic krill season,can preferably have a maximum fat content of about 15%, a minimumprotein content of about 60%, and a maximum moisture content of about10%.

In some embodiments, krill oils obtained with the procedure disclosed inthe present invention can be stabilized by the use of antioxidantsand/or preservatives and/or with a nitrogen-barred layer. Moreover, suchkrill oils can be stored within plastic or metal containers, necessarilysuitable for food-grade, pharmaceutical-grade and/or cosmetic/gradeapplications, in special in stainless steel containers, at roomtemperature or refrigerated, suitably protected from light.

In some embodiments, the present invention provides uses of krill oilsfor preparing krill oil compositions for being used as a dietarysupplement and/or nutraceutical product. This invention also disclosespharmaceutical compositions comprising an effective amount of krill oiland at least one pharmaceutically acceptable transporter, excipient,stabilizer, diluents and/or adjuvant. In some of the embodiments, saidkrill oil compositions are suitable as photoprotectors. Saidphotoprotectors can be formulated as tanning creams and/or tanning oils.In some of the embodiments, said krill oil can be used to enhancecosmetic products. Said cosmetic products are, but no limited to,moisture creams, powder make ups, powder eye shadows, cream eye shadowscompact powders and lipsticks, In some of the embodiments, said krilloil compositions can be effectively used for decreasing cholesterolplasma levels, inhibiting platelet adhesion, inhibiting artery plaqueformation, preventing hypertension, controlling arthritis symptoms,preventing skin cancer, enhancing transdermal transport, reducing thesymptoms of premenstrual symptoms or controlling blood glucose levels ina patient. Furthermore, in some embodiments, nutraceuticals,pharmaceuticals and cosmetics comprising the phospholipids-enrichedkrill oil are also embraced by the present invention.

EXAMPLES

The present invention will be described in more detail using examples.It should be understood that the present invention is not limited by thefollowing Examples.

Example 1 Process to Obtain Krill Oils and Low Fat Krill Meal of thePresent Invention

The description represents an example of the process of the presentinvention. In FIG. 2 there is depicted a flow diagram of this process.The process clearly does not involve the use of organic solvents and/orsupercritical CO₂ fluid.

Antarctic krill was captured during the period of March to May,preferably during the month of May, within krill's fatty period in theOrkney Islands fishing ground, using a pumping catch system, the Krillarriving alive on board a factory vessel and being immediatelyprocessed. Table No 5 shows the fresh raw whole krill composition used.

TABLE 5 Compounds Value Moisture (%) 77.2 Proteins (%) 13.7 Lipids (%)5.3 Ash (%) 3.0 Astaxanthin (mg/Kg) 47

Whole krill was collected into bunkers to drain seawater and transportedon belt conveyors to tanks from wherein it was pumped to the cooker,model Stord Disc Cooker RPH-60, using a screw pump. The continuouscooker with screw conveyor used indirect steam heating (of the cooker)and live steam injection (i.e. direct steam contact with the krill) toincrease the krill temperature from about 0° C. to 93-95° C. The cookerscrew conveyor speed was set at 7-8 rpm.

The cooked krill was then pumped to a Westfalia CD536 two-phase decanterusing a screw pump. The continuous working cooker with screw conveyorused indirect steam heating and live steam to increase the krilltemperature from about 0° C. to 93-95° C. The cooker screw conveyorspeed was set at 7-8 rpm. The cooked krill was pumped to a two-phasedecanter. The heating is done as a single continuous step to bring thekrill up to and thereafter be maintained at the desired temperature >90C, unlike prior art techniques which have multiple heating and coolingsteps.

The two-phase decanter operated at 3,100 rpm and a 2.0 kNm torque wasused, resulting in a decanter liquid phase and a decanter solid phase.Table No 1 shows the decanter liquid and decanter solid composition.

The decanter liquid phase was collected into tanks at a temperature of93-96° C., from wherein it was pumped with a screw pump to a WestfaiaSA100 separator centrifuge at a speed set at 4,600 rpm and immediatelythereafter passed to a purifier separator at a speed set at 6,100 rpm,for obtaining a krill oil with neutral lipids without phospholipids.Example 3 below shows this neutral lipids oil composition andcharacteristics.

The decanter solid phase was pressed in a Stord Twin screw pressresulting in a press liquid phase and press cake phase. Table No 6 showsthe composition obtained from these two phases.

TABLE 6 Compounds Press Liquid (%) Press Cake (%) Moisture 77.7 54.5Proteins 6.1 32.7 Lipids 14.0 6.0 Ash 0.9 4.6

The press cake was dried until it reached a moisture content lower than10% using a Atlas Stord Disc Dryer (rotaplate dryer). Example 5 showsthe krill meal composition obtained from this test.

The press liquid at a temperature of 93-96° C. was pumped with a screwpump to a specific separator centrifuge at a speed set at 4,600 rpm andimmediately thereafter to a specific purifier separator at a speed set6,100 rpm, to separate the krill oil with phospholipids. Example 4,below, shows the composition characteristics of this krill oil with DHAand EPA linked to the phospholipids fraction obtained from the presentexperiment.

Example 2 Flow Diagram and Mass Balance of the Process to Obtain KrillOils and Low Fat Krill Meal of the Present Invention

This example shows an estimate mass balance of a production line for theproduct of the present invention when raw krill is within the seasonwhen fat content is high (estimated at 5%) although not necessarily thehighest fat content found in raw South Antarctic Krill and without theaddition or recovery of stick water.

The following tables are an estimated mass balance of the process of thepresent invention without stick water recovery, utilizing raw krill withaverage-high fat content (around 5% w/w).

TABLE 7 Product MT/Hr Yield (%) Krill Meal (without 1.63 16.3concentrate) Krill Oil with Phospholipids 0.22 2.2 Krill Oil withNeutral Lipids 0.07 0.7 Total 1.92 19.2

TABLE 8 FRESH RAW WHOLE KRILL Kg/Hr (%) Kg/Hr (%) DS 1,800 18.0 F 5005.0 Phospholipids 200 40.0 Neutral Lipids 295 59.0 Cholesterol 5 1.0 M7,700 77.0 Total 10,000 100 500 100

TABLE 9 DECANTER SOLIDS Kg/Hr (%) Kg/Hr (%) DS 1,386 28.7 F 401 8.3Phospholipids 190 47.4 Neutral Lipids 208 51.9 Cholesterol 3 0.7 M 3,04363.0 Total 4,830 100 401 100

TABLE 10 PRESS CAKE Kg/Hr (%) Kg/Hr (%) DS 1,248 40.8 F 160 5.2Phospholipids 104 65.0 Neutral Lipids 54 34.0 Cholesterol 2 1.0 M 1,65254.0 Total 3,060 100 160 100

TABLE 11 PRESS CAKE + SLUDGE Kg/Hr (%) Kg/Hr (%) DS 1,303 35.6 F 195 5.3Phospholipids 121 62.4 Neutral Lipids 70 35.9 Cholesterol 3 1.7 M 2,16259.1 Total 3,660 100 195 100

TABLE 12 KRILL SOLUBLES Kg/Hr (%) DS 0 0.0 F 0 0.0 M 0 0.0 Total 0 0.0

TABLE 13 FEED TO DRIERS RCD Kg/Hr (%) Kg/Hr (%) DS 1,303 35.6 F 195 5.3Phospholipids 121 62.4 Neutral Lipids 70 35.9 Cholesterol 3 1.7 M 2,16259.1 Total 3,660 100 195 100

TABLE 14 KRILL MEAL Kg/Hr (%) Kg/Hr (%) DS 1,303 80.0 F 195 12.0Phospholipids 121 62.4 Neutral Lipids 70 35.9 Cholesterol 3 1.7 M 1308.0 Total 1,628 100 195 100

TABLE 15 EVAPORATOR Kg/Hr 0

TABLE 16 EVAPORATED DRIERS RCD Kg/Hr 2,032

TABLE 17 DECANTER LIQUID Kg/Hr (%) Kg/Hr (%) DS 414 8.0 F 99 1.9Phospholipids 10 10.1 Neutral Lipids 87 87.9 Cholesterol 2 2.0 M 4,65790.1 Total 5,170 100 99 100

TABLE 18 OIL WITH NEUTRAL LIPIDS Kg/Hr (%) Kg/Hr (%) Oil 69 100Phospholipids 0 0 Neutral Lipids 68 98.6 Cholesterol 1 1.4 Total 69 10069 100

TABLE 19 PRESS LIQUID Kg/Hr (%) Kg/Hr (%) DS 139 7.8 F 241 13.6Phospholipids 86 35.7 Neutral Lipids 153 63.7 Cholesterol 1 0.6 M 1,39178.6 Total 1,770 100 241 100

TABLE 20 OIL WITH PHOSPHOLIPIDS Kg/Hr (%) Kg/Hr (%) Oil 220 100Phospholipids 79 35.7 Neutral Lipids 140 63.7 Cholesterol 1 0.3 Total220 100 219 100

TABLE 21 SLUDGE 1 Kg/Hr (%) Kg/Hr (%) DS 41 10.5 F 18 4.5 Phospholipids10 56.1 Neutral Lipids 7 38.3 Cholesterol 1 5.6 M 336 85.0 Total 395 10018 100

TABLE 22 SLUDGE 2 Kg/Hr (%) Kg/Hr (%) DS 14 6.8 F 17 8.2 Phospholipids 744.2 Neutral Lipids 9 51.7 Cholesterol 1 4.2 M 174 85.0 Total 205 100 17100

TABLE 23 STICK WATER 1 Kg/Hr (%) Kg/Hr (%) DS 372 7.9 F 12 0.3Phospholipids 0 0 Neutral Lipids 12 100 Cholesterol 0 0 M 4,322 91.8Total 4,706 100 12 100

TABLE 24 STICK WATER 2 Kg/Hr (%) Kg/Hr (%) DS 125 9.3 F 4 0.3Phospholipids 0 0 Neutral Lipids 5 100 Cholesterol 0 0 M 1,216 90.4Total 1,345 100 5 100

DS=Dry Solids Content F=Fat Content M=Moisture Content MT=Metric Ton

The data from the above tables show that the inventive process allowsmore fat to found in the liquid portion and thus allows easier recovery.See Table 19 in particular with regard to the level of phospholipids inthe press liquid and Table 17 with regard to the level of triglyceridesin the decanted liquid.

The following tables shows an estimate mass balance of a production linefor the product of the present invention when raw krill is within theseason when fat content is high (estimated at 5%) although notnecessarily the highest fat content found in raw South Antarctic Krilland with the addition or recovery of stick water.

TABLE 25 Production MT/Hr Yield (%) Krill Meal (with concentrate) 2.1921.9 Krill Oil with Phospholipids 0.22 2.2 Krill Oil with Neutral Lipids0.07 0.7 Total 2.47 24.7

FRESH RAW WHOLE KRILL Kg/Hr (%) Kg/Hr (%) DS 1,800 18.0 F 500 5.0Phospholipids 200 40.0 Neutral Lipids 295 59.0 Cholesterol 5 1.0 M 7,70077.0 Total 10,000 100 500 100

TABLE 27 DECANTER SOLIDS Kg/Hr (%) Kg/Hr (%) DS 1,386 28.7 F 401 8.3Phospholipids 190 47.4 Neutral Lipids 208 51.9 Cholesterol 3 0.7 M 3,04363.0 Total 4,830 100 401 100

TABLE 28 PRESS CAKE Kg/Hr (%) Kg/Hr (%) DS 1,248 40.8 F 160 5.2Phospholipids 104 65.0 Neutral Lipids 54 34.0 Cholesterol 2 1.0 M 1,65254.0 Total 3,060 100 160 100

TABLE 29 PRESS CAKE + SLUDGE Kg/Hr (%) Kg/Hr (%) DS 1,303 35.6 F 195 5.3Phospholipids 121 62.4 Neutral Lipids 70 36.1 Cholesterol 3 1.5 M 2,16259.1 Total 3,660 100 195 100

TABLE 30 KRILL SOLUBLES Kg/Hr (%) Kg/Hr (%) DS 497 48.5 F 16 1.5Phospholipids 0 0 Neutral Lipids 16 100 Cholesterol 0 0 M 513 50.0 Total1,026 100 16 100

TABLE 31 FEED TO DRIERS RCD Kg/Hr (%) Kg/Hr (%) DS 1,800 38.4 F 211 4.5Phospholipids 121 57.6 Neutral Lipids 86 41.0 Cholesterol 3 1.4 M 2,67557.1 Total 4,686 100 211 100

TABLE 32 KRILL MEAL Kg/Hr (%) Kg/Hr (%) DS 1,800 82.4 F 211 9.6Phospholipids 121 57.6 Neutral Lipids 86 41.0 Cholesterol 3 1.4 M 1758.0 Total 2,185 100 211 100

TABLE 33 EVAPORATOR Kg/Hr 5,025

TABLE 34 EVAPORATED DRIERS RCD Kg/Hr 2,500

TABLE 35 DECANTER LIQUID Kg/Hr (%) Kg/Hr (%) DS 414 8.0 F 99 1.9Phospholipids 10 10.1 Neutral Lipids 87 87.9 Cholesterol 2 2.0 M 4,65790.1 Total 5,170 100 99 100

TABLE 36 OIL WITH NEUTRAL LIPIDS Kg/Hr (%) Kg/Hr (%) Oil 69 100Phospholipids 0 0 Neutral Lipids 68 98.6 Cholesterol 1 1.4 Total 69 10069 100

TABLE 37 PRESS LIQUID Kg/Hr (%) Kg/Hr (%) DS 139 7.8 F 241 13.6Phospholipids 86 35.7 Neutral Lipids 153 63.7 Cholesterol 1 0.6 M 1,39178.6 Total 1,770 100 241 100

TABLE 38 OIL WITH PHOSPHOLIPIDS Kg/Hr (%) Kg/Hr (%) Oil 220 100Phospholipids 79 35.7 Neutral Lipids 140 63.7 Cholesterol 1 0.5 Total220 100 220 100

TABLE 39 SLUDGE 1 Kg/Hr (%) Kg/Hr (%) DS 41 10.5 F 18 4.5 Phospholipids10 56.1 Neutral Lipids 7 38.3 Cholesterol 1 5.6 M 336 85.0 Total 395 10018 100

TABLE 40 SLUDGE 2 Kg/Hr (%) Kg/Hr (%) DS 14 6.8 F 17 8.2 Phospholipids 744.2 Neutral Lipids 9 53.5 Cholesterol 0 2.4 M 174 85.0 Total 205 100 17100

TABLE 41 STICK WATER 1 Kg/Hr (%) Kg/Hr (%) DS 372 7.9 F 12 0.3Phospholipids 0 0 Neutral Lipids 12 100 Cholesterol 0 0 M 4,322 91.8Total 4,706 100 12 100

TABLE 42 STICK WATER 2 Kg/Hr (%) Kg/Hr (%) DS 125 9.3 F 4 0.3Phospholipids 0 0 Neutral Lipids 4 100 Cholesterol 0 0 M 1,216 90.4Total 1,345 100 4 100

Example 3 Composition of the Neutral Lipid Enriched Krill Oil of thePresent Invention

TABLE 43 Neutral Lipids % w/w Triglycerides 84.6 Diglycerides 4.9 FreeFatty Acids ND Monoglycerides ND Total 89.5 Phospholipids ND <0.5alpha-Tocopherol 0.7 Fatty acid FAME (Fatty Acid Analysis Methyl Ester)% w/w Total Sample  8:0 0.0  9:0 0.0 10:0 0.0 11:0 0.0 12:0 0.3 13:0 0.014:0 16.3 15:0 0.5 16:0 18.2 17:0 0.3 18:0 1.5 19:0 1.0 20:0 0.0 22:00.1 23:0 0.0 24:0 0.0 Saturated Total 38.3 11:1 0.0 13:1 0.0 14:1 0.216:1 9.5 17:1 1.2 18:1 cis 14.6 18:1 trans 6.4 20:1 1.2 22:1 0.1 24:10.0 Monounsaturated Total 33.2 18:2 1.3 18:3 (6, 9, 12) 0.2 18:3 (9, 12,15) 0.6 20:2 0.0 20:3 (8, 11, 14) 0.0 20:4 0.0 20:3 (5, 8, 11) 0.2 20:53.3 22:2 0.2 22:3 0.0 22:4 0.0 22:5 N3 0.1 22:6 1.1 PolyunsaturatedTotal 7.1 Total All 78.6 ND = Not Detected

Example 4 Composition of the Phospholipids Enriched Krill Oil of thePresent Invention

TABLE 44 Composition (% total lipid)* Preferred ranges +/− Composition20% for each value or (% total lipid)* Alternatively +/− More preferredLipid Class 10% for each value ranges Sterol esters 1.2   1-1.5Triacylglycerols 45.6 40-50 Free fatty acids 3.4 2-4 Cholesterol/sterols4.3 3-5 Diacylglycerols** 7.7  5-10 Monoacylglycerols 0.9 0.2-1.5 Totalneutral lipids 63.1 Phosphatidylcholine 25.2 23-29Phosphatidylethanolamine 7.3 6-9 Phosphatidylserine 1.5 1.2-4.1Phosphatidylinositol 1.3 0.9-2.2 Phosphatidylglycerol + 1.0 0.6-1.4cardiolipin Lysophosphatidylcholine 0.6   0-1.2 Total polar lipids 36.9Values are means of duplicate analyses *, as determined byHPTLC/densitometry. **, contains pigments.

The low levels of lysophosphatidylcholine (LPC) found in compositionsmade in accordance with the processes of the invention are noteworthy.Lysophosphatidylcholine (LPC) is the resulting compound of thedecomposition of Phosphatidylcholine (PC) throughout the entireextraction process and/or raw frozen krill storage normally through anextended period of time. The lower the content of LPC is the result ofprocessing krill very fresh and limited decomposition. Results of aprocess that works with raw material that has not gone through differentdeterioration steps. This low LPC content is a very distinctiveparameter of our invention as we are working with extremely fresh rawmaterial, processed on board from fresh raw krill in the same fishingground where it is captured. Other processes extract the oil on shore,from a raw material that has been previously pre-processed on board (butlater re-processed on shore) Prior art methods result in PL oils withLPC above 5%.

Fatty Acid Composition of Total Lipid

TABLE 45 % total fatty acids Preferred ranges +/− 20% for each valueMore Preferred ranges +/− 10% for each value 14:0 12.55 br 15:0 0.2815:0 0.48 br 16:0 0.13 16:0 21.67 br 17:0 0.43 17:0 0.10 18:0 1.47 20:00.29 Total saturated 37.39 16:1n-9 0.38 16:1n-7 6.81 16:1 isomer 0.6017:1n-8 0.29 18:1n-9 12.21 18:1n-7 7.43 20:1n-11 0.07 20:1n-9 0.9320:1n-7 0.42 22:1n-11 0.53 22:1n-9 0.14 24:1n.9 0.21 Totalmonounsaturated 30.02 18:2n-6 1.74 18:3n-6 0.17 20:2n-6 0.08 20:3n-60.10 20.4n-6 0.33 Total n-6 PUFA (Polyunsaturated 2.43 Fatty Acids)18:3n-3 0.98 18:4n-3 3.24 20:3n-3 0.10 20:4n-3 0.34 20:5n-3 15.2022:5n-3 0.40 22:6n-3 7.55 Total n-3 PUFA 27.81 16:2 0.69 16:3 0.27 16:41.38 Total 16C PUFA 2.35 TOTAL PUFA 32.59 Total 100.00 Values are meansof duplicate analyses Limit of quantification (LOQ) for fatty acidanalysis = 0.06%

Fatty Acid Composition of Phosphatidylcholine/Lysophosphatidylcholine

TABLE 46 % total fatty acids Preferred ranges +/− 20% for each valueMore Preferred ranges +/− 10% for each value 14:0 2.79 br 15:0 0.10 15:00.37 br 16:0 0.12 16:0 27.17 br 17:0 0.47 17:0 0.15 18:0 1.04 20:0 0.22Total saturated 32.44 16:1n-9 0.15 16:1n-7 1.73 16:1 isomer 0.28 17:1n-80.14 18:1n-9 4.46 18:1n-7 5.14 20:1n-11 <LOQ 20:1n-9 0.60 20:1n-7 0.2722:1n-11 1.12 22:1n-9 0.50 24:1n.9 0.46 Total monounsaturated 14.8718:2n-6 1.55 18:3n-6 0.27 20:2n-6 <LOQ 20:3n-6 0.10 20.4n-6 0.59 Totaln-6 PUFA 2.50 18:3n-3 1.05 18:4n-3 1.98 20:3n-3 0.14 20:4n-3 0.4720:5n-3 31.50 22:5n-3 0.63 22:6n-3 14.09 Total n-3 PUFA 49.87 16:2 0.1016:3 0.12 16:4 0.11 Total 16C PUFA 0.33 TOTAL PUFA 52.70 Total 100.00Values are means of duplicate analyses Limit of quantification (LOQ) forfatty acid analysis = 0.06%

Fatty Acid Composition of Phosphatidylethanolamine

TABLE 47 % total fatty acids Preferred ranges 30/− 20% for each valueMore Preferred ranges 30/− 10% for each value 14:0 0.56 br 15:0 0.0715:0 0.20 br 16:0 0.17 16:0 14.65 br 17:0 0.39 17:0 0.83 18:0 1.24 20:0<LOQ Total saturated 18.11 16:1n-9 0.25 16:1n-7 0.61 16:1 isomer 0.2717:1n-8 <LOQ 18:1n-9 3.92 18:1n-7 14.03 20:ln-11 <LOQ 20:1n-9 0.4820:1n-7 0.10 22:1n-11 <LOQ 22:1n-9 <LOQ 24:1n.9 0.08 Totalmonounsaturated 19.79 18:2n-6 0.95 18:3n-6 0.49 20:2n-6 0.15 20:3n-60.15 20.4n-6 1.35 Total n-6 PUFA 3.10 18:3n-3 0.38 18:4n-3 0.25 20:3n-30.14 20:4n-3 0.42 20:5n-3 23.35 22:5n-3 0.75 22:6n-3 33.12 Total n-3PUFA 58.43 16:2 0.14 16:3 0.17 16:4 0.27 Total 16C PUFA 0.58 TOTAL PUFA62.10 Total 100.00 Values are means of duplicate analyses Limit ofquantification (LOQ) for fatty acid analysis = 0.06%

Fatty Acid Composition of Triacylglycerol

TABLE 48 % total fatty acids Preferred ranges +30/- 20% for each valueMore Preferred ranges +30/- 10% for each value 14:0 20.52 br 15:0 0.4215:0 0.61 br 16:0 0.15 16:0 21.85 br 17:0 0.48 17:0 0.09 18:0 1.67 20:00.31 Total saturated 46.11 16:1n-9 0.21 16:1n-7 9.90 16:1 isomer 0.7217:1n-8 0.36 18:1n-9 16.76 18:1n-7 8.26 20:ln-11 0.08 20:1n-9 1.1420:1n-7 0.55 22:1n-11 0.24 22:1n-9 0.11 24: ln.9 <LOQ Totalmonounsaturated 38.32 18:2n-6 1.79 18:3n-6 0.22 20:2n-6 <LOQ 20:3n-6<LOQ 20.4n-6 0.08 Total n-6 PUFA 2.09 18:3n-3 0.86 18:4n-3 3.40 20:3n-30.07 20:4n-3 0.17 20:5n-3 4.05 22:5n-3 0.20 22:6n-3 1.49 Total n-3 PUFA10.22 16:2 1.00 16:3 0.37 16:4 1.88 Total 16C PUFA 3.25 TOTAL PUFA 15.57Total 100.00 Values are means of duplicate analyses Limit ofquantification (LOQ) for fatty acid analysis = 0.06%

Lipid class composition of lipid samples and fatty acid compositions ofindividual lipids were determined by high performance thin-layerchromatography (HPTLC) and quantitation using a scanning densitometryaccording to Henderson and Tocher (Henderson, R. J. and Tocher, D. R.(1992) Thin-layer chromatography. In Lipid Analysis: A PracticalApproach (Hamilton, R. J., and Hamilton, S., eds.) pp. 65-111, OxfordUniversity Press, Oxford).

Example 5 Other Characteristics of the Phospholipid Enriched Krill Oilof the Present Invention

TABLE 49 Composition Preferred ranges +/− 20% for each value MorePreferred ranges +/− 10% for each value Vitamin E Composition (*) 13.80in mg/Kg Peroxide Value (*) in Meq/Kg 0.0 Iodine Value (IV) (**) (g/100g) 150.37 (*)Calculated in Duplicate (**) Calculated by the modificationof Ham et al. (J.A.O.C.S. 75, 1445-1446(1998)) of the AOCS recommendedpractice Cd 1c-85.

Example 6 Heavy Metals Content for the Phospholipid Enriched Krill Oilof the Present Invention

TABLE 50 Present Neptune 999 Invention Bioressouces (Triple Nine) HeavyMetal %/w %/w %/w Antimony ppm <0.02 <0.03 <0.02 Arsenic ppm <0.05 2.314.49 Bismuth ppm <0.02 0.17 <0.02 Cadmium ppm <0.02 <0.03 0.07 Copperppm <0.04 0.21 13.1 Lead ppm <0.02 0.09 0.27 Mercury ppm <0.02 <0.03<0.04 Molybdenum ppm <0.02 <0.03 0.06 Silver ppm <0.02 <0.03 0.45 Tinppm <0.02 <0.03 0.05 Total ppm <0.05 2.78 18.49

Example 7 Composition of the Low Fat Krill Meal of the Present Inventwith Krill Captured During South Antarctic's Krill Fatty Period

TABLE 51 Compounds Value Moisture (%) 8.0 Proteins (%) 66.1 Lipids (%)12.1 Ash (%) 9.3 Astaxanthin (mg/Kg) 119

Example 8 Dietary Supplement (Nutraceutical/Dietary) Based on Krill Oil

It is described a food rich in fat material for complementing essentialfatty acids. Such food was formulated in cookie form elaborated usingkrill oil, krill meal or krill dried complex.

In a bowl 400 g Quaker oats was mixed with 100 g of flour, 500 g ofsugar, 1 egg, 150 mL of krill oil of this invention and 10 mL of vanillaextract. Once completely homogenized, the cookies were molded weighingeach 25 g, and were baked at 160° C. for 15 min. as longer cooking timesdestroys the astaxanthin.

The amount of cookies used in the diets will depend on the amount ofessential fatty acids such as 18:2 and 18:3 required, and the necessarycalories.

Example 9 Photoprotector

The krill oil prepared as described in the examples 3 and 4 can be usedfor the preparation of tanning creams and tanning oils for solarprotection.

A) Tanning Creams.

In this example are described two tanning creams, one with solarprotection factor 5 (SPF5) and one with solar protection factor 20(SPF20).

TABLE 52 Composition per 100 g of Tanning Cream Component SPF 5 SPF 20Krill Oil 5.00 g 5.00 g Carbopol 940 0.25 g 0.25 g Cetilic alcohol 0.50g 0.50 g Methyl paraben 0.15 g 0.15 g Estearic acid III pr. 2.00 g 2.00g Propyl paraben 0.15 g 0.15 g Panalen 5.00 g 5.00 g EDTA disodium 0.10g 0.10 g Triethanolamine 0.50 g 0.50 g Propylenglycol 2.50 g 2.50 gOctylmethoxycinamate — 7.50 g Benzophenone 3 — 3.00 g Titanium dioxide —0.50 g Imidazolidinilurea 0,30 g 0.30 g Glyceril monoestearate 1.00 g1.00 g Natural essence c.s. c.s. Isopropyl miristate 2.00 g 2.00 g Waterc.s. c.s.

B) Tanning Oil:

The following description corresponds to tanning oil that contains krilloil as the unique sun-blocking agent.

TABLE 53 Composition per Compound 100 g of Tanning Oil Cosmetic liquidVaseline 80.0 g Hydrogenated polybutenes  5.0 g Krill oil 14.7 g Essence 0.3 g

Example 10 Cosmetic Products Based on Krill Oil

Since krill oil has several biological activities, such as being apigmentant, antioxidant capacity, EPA and essential fatty acids content,it is possible to design cosmetic products.

A) Moisturizing Cream.

TABLE 54 Composition per Compound 100 g of Moisturizing Cream Estearicacid 1.0 g Anionic glyceril monoestearate A.E. 0.7 g Neutral glycerilmonoesteararte 0.5 g Hydrogenated polybutenes 3.2 g Krill oil 10.0 g Propylenglycol 2.0 g Isopropyl miristate 1.5 g Carboxyvinyl polymer 0.3g Propylparabene 0.1 g Methylparabene 0.1 g Essence 0.3 g EDTA disodium0.2 g Triethanolamine 99% 1.2 g Demineralized water 78.9 g 

B) Powder Makeup.

In this example it is described a base formula for the elaboration ofpowder makeup that contains 10% (w/w) krill oil.

TABLE 55 Composition per Compound 100 g of Makeup Talcum powder 52.56 gKrill oil 10.00 g Mica: Titanuim dioxide (2:1) 26.00 g Magnesiumestearate  3.50 g Isopropyl miristate  1.60 g Oleic alcohol  2.60 gOctylpalmitate  3.40 g Methylparabene  0.17 g Propylparabene  0.17 g

C) Powder Eye Shadow.

In this example it is described a base formula for the elaboration of apowder eye-shadow that contains 10% (w/w) krill oil of this invention.

TABLE 56 Composition per Compound 100 g of Eye Shadow Talcum powder51.46 g Krill oil 10.00 g Mica: Titanium dioxide (3:1) 27.00 g Magnesiumestearate  3.50 g Isopropyl miristate  1.60 g Oleic alcohol  2.60 gOctylpalmitate  3.50 g Methylparabene  0.17 g Propylparabene  0.17 g

D) Cream Eye Shadow

In this example, a base formula for the elaboration of a cream eyeshadow that contains 5.7% (w/w) krill oil of this invention isdescribed.

TABLE 57 Composition per Compound 100 g of Eye Shadow Talcum powder  4.0g Estearic acid  9.5 g Isoestearic acid  1.9 g Krill Oil  5.7 g Titaniumdioxide  1.9 g Aluminum and magnesium  3.6 g silicate Propylenglycol16.1 g Triethanolamine 11.0 g Methylparabene  0.2 g Propylparabene  0.2g Deionized water 45.9 g

E) Compact Powder.

In this example, a base formula for the elaboration of a compact powderthat contains 10% (w/w) krill oil of the present invention is described.

TABLE 58 Composition per Compound 100 g of Compact Powder Kaolin 32.0 gKrill Oil 10.0 g Mica: Titanium dioxide (2:1)  1.6 g Magnesium estearate 4.1 g Octylpalmitate  2.5 g Propylparabene  0.5 g Talcum powder 49.3 g

F) Lipstick.

In this example, a base formula for a lipstick that contains 3% (w/w)krill oil is described.

TABLE 59 Composition per Compound 100 g of Lipstick Castor Oil 42.04 gKrill Oil  3.00 g Oleic alcohol  2.60 g Lanolin 25.00 g Sorbitanmonoestearate  1.30 g Ozokerite  4.50 g Carnauba wax  5.20 g Beeswax 5.60 g Estearic acid  4.90 g Candle wax  5.60 g Methylparabene  0.13 gPropylparabene  0.13 g

Example 11 Pharmaceutical Products Based on Krill Oil

Krill and/or marine oil has been shown to decrease cholesterol in vivo.It also inhibits platelet adhesion and plaque formation and reducesvascular endothelial inflammation in a patient. It can offerhypertension prophylaxis. It prevents oxidation of low-densitylipoprotein. It may have an inhibitory effect on the secretion of VLDLdue to increased intracellular degradation of apo B-100. It also offersa post-myocardial infarction prophylaxis because of its ability todecrease CIII apolipoprotein B, to decrease CIII non-apolipoprotein Blipoproteins and to increase antithrombin III levels. Krill and/ormarine oil is suitable for prophylactic usage against cardiovasculardisease in human where cardiovascular disease relates to coronary arterydisease, hyperlipidemia, hypertension, ischemic disease (relating toangina, myocardial infarction, cerebral ischemia, shock without clinicalor laboratory evidence of ischemia, arrhythmia).

A pharmaceutical composition of krill oil of this invention comprisescapsules containing 1 mL of krill oil described in examples 3 and 4. Apharmaceutical composition of krill dried complex of this inventioncomprises capsules containing 1 to 5 g of krill dried complex.

What we claim is:
 1. A solid krill product produced by squeezing thefirst solid krill fraction resulting from an organic solvent-freeprocess for producing krill oil comprising: a) cooking krill in a cookervessel for a time and at a temperature sufficient to denature theprotein content of the krill and cause a first solid krill fraction anda first liquid krill fraction to be formed while substantially avoidingemulsification of the first solid and first liquid krill fractions; b)removing the first solid and first liquid krill fractions from thecooker vessel at a temperature of at least about 90° C.; c) separatingthe first solid fraction and the first liquid fraction; and d) obtainingkrill oil from either the first liquid fraction or the first solidfraction, said separating and said obtaining steps being carried outwithout the use of organic solvents.
 2. A liquid krill oil containingproduct produced by recovering the first liquid krill fraction resultingfrom an organic solvent-free process for producing krill oil comprising:a) cooking krill in a cooker vessel for a time and at a temperaturesufficient to denature the protein content of the krill and cause afirst solid krill fraction and a first liquid krill fraction to beformed while substantially avoiding emulsification of the first solidand first liquid krill fractions; b) removing the first solid and firstliquid krill fractions from the cooker vessel at a temperature of atleast about 90° C.; c) separating the first solid fraction and the firstliquid fraction; and d) obtaining krill oil from either the first liquidfraction or the first solid fraction, said separating and said obtainingsteps being carried out without the use of organic solvents.
 3. A liquidkrill oil containing product produced by squeezing the first solid krillfraction resulting from an organic solvent-free process for producingkrill oil comprising: a) cooking krill in a cooker vessel for a time andat a temperature sufficient to denature the protein content of the krilland cause a first solid krill fraction and a first liquid krill fractionto be formed while substantially avoiding emulsification of the firstsolid and first liquid krill fractions; b) removing the first solid andfirst liquid krill fractions from the cooker vessel at a temperature ofat least about 90° C.; c) separating the first solid fraction and thefirst liquid fraction; and d) obtaining krill oil from either the firstliquid fraction or the first solid fraction, said separating and saidobtaining steps being carried out without the use of organic solvents.4. A dried solid krill meal obtained by drying the solid krill productobtained by an organic solvent-free process for producing krill oilcomprising: a) cooking krill in a cooker vessel for a time and at atemperature sufficient to denature the protein content of the krill andcause a first solid krill fraction and a first liquid krill fraction tobe formed while substantially avoiding emulsification of the first solidand first liquid krill fractions; b) removing the first solid and firstliquid krill fractions from the cooker vessel at a temperature of atleast about 90° C.; c) separating the first solid fraction and the firstliquid fraction; and d) obtaining krill oil from either the first liquidfraction or the first solid fraction, said separating and said obtainingsteps being carried out without the use of organic solvents, wherein theobtaining krill oil from the first solid fraction comprises squeezingthe first solid fraction to obtain a second solid fraction and a secondliquid fraction; and separating the second liquid fraction therefrom toobtain krill oil enriched with phospholipids and water.
 5. A compositioncontaining krill oil enriched in neutral lipids, comprising: neutrallipids in an amount of about 50 to about 100% by weight; DHA and EPA inan amount of about 2 to about 45% by weight; phospholipids in an amountof less than about 10% by weight; astaxanthin in an amount of about 200to about 1,500 mg/kg; and free fatty acid content in an amount of about3.4% or less by weight.
 6. A composition containing krill oil enrichedin phospholipids comprising: phospholipids in an amount of about 30 toabout 70% by weight; DHA and EPA in an amount of about 10 to about 70%by weight; neutral lipids in an amount of about 30 to about 70% byweight; astaxanthin in an amount of about 200 to about 1,500 mg/kg;phosphatidylserine in an amount of about 1.5 or more by weight totallipids; lysophosphatidylcholine in an amount of about 0.6% or less byweight total lipids; and free fatty acids in an amount of about 3.4% orless by weight total lipids.
 7. A phospholipid-enriched krill oilcomposition containing reduced heavy metal content, comprising krill oiland Heavy Metal %/wt Antimony Ppm <0.02 Arsenic Ppm <0.05 Bismuth Ppm<0.02 Cadmium Ppm <0.02 Copper Ppm <0.04 Lead Ppm <0.02 Mercury Ppm<0.02 Molybdenum Ppm <0.02 Silver Ppm <0.02 Tin Ppm <0.02 Total Ppm<0.05